One of the major hallmarks of Alzheimer's disease (AD) is cerebral depositions of the beta-amyloid protein (AB) which is derived from a larger beta-amyloid precursor proteins (APP). Neurochemically, AD is characterized by a reduction in the presynaptic markers of the cholinergic system particularly in the areas of the brain related to memory and learning. This proposal addresses the processing of APP and how it is affected by a selected group of cholinesterase inhibitors (ChE-Is), specially the chemical tacrine. The primary clinical effect of tacrine is by its inhibition of the enzyme cholinesterase (ChE), thus resulting in a general increased level of acetylcholine whose levels appear to be reduced in A.D. However, this treatment has only moderate benefits in a limited number of patients. The cholinergic effect of tacrine may improve the synapses, while another action of the drug (e.g., lysosomotropic property) could be different. Our proposal will attempt to demonstrate non-cholinergic functions of tacrine using a cell culture model. It is based on the finding that treatment with tacrine markedly suppressed secretion of APP in cultured cells. Hypotheses are: i) noncholinergic functions of tacrine are independent of its well known anti-ChE activity, and ii) the lysosomotropic function of tacrine can be used to study the processing of APP. The specific aims are: l) to investigate the effect of ChE-Is on the secretion of APP and 2) to determine the effect of the selected ChE-Is (from S.A.1) on the carboxyl-truncated fragments (CTFs) of APP. Experiments will be performed in neuronal and astrocytic cell lines in the presence of tacrine and a selected group of ChE-Is. APP will be analyzed by immunochemicaI and molecular biological techniques. Effects of ChE-Is will also be studied in neuroblastoma cells transfected with either wild type APP751 or mutants of APP751 identified in familial AD (FAD). As control, we will study the effects of ChE-Is on other proteins such as heat-shock proteins, protease nexin- l and synaptophysin. Possible interpretations of the outcomes of this proposal are as follows: if the effect of tacrine is solely on ChE then one would expect the same effect of APP secretion to be seen with other ChE-Is. If the effect of tacrine is on the intracellular processing of APP, for instance due to the drug's lysosomotropic property then it would suggest future potential therapies should be focused on that feature of tacrine rather than on its ChE activity only. If the drug has unique effects on processing of APP, that would suggest tacrine or one of its derivatives might be further investigated and or developed to maximize potential effects in decreasing amyloid depositions.